Endothelial cells, normally present as a monolayer in the intimal layer of the arterial wall, are believed to play an important role in the regulation of smooth muscle cell (SMC) proliferation in vivo. Endothelial cells are seriously disrupted by most forms of vascular injury, including that caused by percutaneous transluminal coronary angioplasty and similar procedures. Approximately 35–50% of patients treated by percutaneous transluminal coronary angioplasty experience clinically significant renarrowing of the artery, or restenosis, within six months of the initial treatment. Restenosis is due, at least in part, to migration and proliferation of smooth muscle cells in the arterial wall along with increases in secretion of matrix proteins to form an obstructive neointimal layer within the arterial wall. Similar issues limit the performance of vascular grafts. The processes that regulate arterial wound healing following vascular injury, such as that caused by angioplasty, are as yet poorly understood, but are believed to involve a complex cascade of blood and vessel wall-derived factors.
Numerous factors that stimulate intimal thickening and restenosis have been identified through administration of exogenous proteins, genetic alteration of cells, or through the blockade of certain signals using antibodies or other specific growth factor inhibitors. These smooth muscle cell mitogens and chemoattractants derive from both the blood or thrombus formation and from the vessel wall itself. Endothelial cells produce a number of substances known to down-regulate smooth muscle cell proliferation, including heparin sulfate, prostacyclin (PG12), and NO.
NO is an endothelium-derived target molecule useful for the prevention of restenosis because, in addition to limiting the proliferation of smooth muscle cells (Garg et al., (1989) J. Clin. Invest., 83:1774–7), NO reduces platelet aggregation (de Graaf et al., (1992) Circulation, 85:2284–90; Radomski et al., (1987) Br. J. Pharmacol., 92:181–7), increases endothelial cell proliferation (Ziche et al., (1993) Biochem. Biophys. Res. Comm., 192:1198–1203), and attenuates leukocyte adhesion (Lefer et al., (1993) Circulation, 88:2337–50), all of which are highly desirable for the reduction of intimal thickening and restenosis (Loscalzo, (1996) Clin. Appl. Thromb. Hemostas., 2:7–10). Because of the complexity of the restenotic process, approaches that act upon multiple targets are the most likely to be successful.
The mechanisms whereby NO affects these multiple responses are not fully understood as yet, but it is known that NO activates soluble guanylate cyclase by binding to its heme moiety, thereby elevating the levels of cyclic guanosine monophosphate (cGMP), an intracellular second messenger with multiple cellular effects (Moro et al., (1996) Proc. Natl. Acad. Sci. USA, 93:1480–5). The effects of NO can often be mimicked by the administration of cGMP or more stable derivatives of cGMP (Garg et al., (1989) J. Clin. Invest., 83:1774–7). In addition, NO has been found to inhibit ribonucleotide reductase, an enzyme that converts ribonucleotides into deoxy ribonucleotides, thus significantly impacting DNA synthesis (Lepoivre et al., (1991) Biochem. Biophys. Res. Comm., 179:442–8; Kwon et al., (1991) J. Exp. Med., 174:761–7), as well as several enzymes involved in cellular respiration (Stuehr et al., (1989) J. Exp. Med., 169:1543–55).
A number of molecules that produce NO under physiological conditions (NO donors) have been identified and evaluated both in vitro and in vivo. NO donor molecules exert biological effects mimicking those of NO and include S-nitrosothiols (Diodati et al, (1993) Thromb. Haem., 70:654–8; Lefer et al., (1993) Circulation, 88:2337–50; DeMeyer et al., (1995) J. Cardiovasc. Pharmacol., 26:272–9), organic nitrates (Ignarro et al., (1981) J. Pharmacol. Exp. Ther., 218:739–49), and complexes of NO with nucleophiles (Diodati et al., (1993) Thromb. Haem., 70:654–8; Diodati et al., (1993) J. Cardiovasc. Pharmacol., 22:287–92; Maragos et al., (1993) Cancer Res., 53:564–8). Most of these have been low molecular weight molecules that are administered systemically and have short half-lives under physiologic conditions, thus exerting effects upon numerous tissue types with a brief period of activity. In addition, L-arginine is often thought of as a NO donor, as L-arginine is a substrate for NO synthase, and thus administration of L-arginine increases endogenous NO production and elicits responses similar to those caused by NO donors in most cases (Cooke et al., (1992) J. Clin. Invest., 90:1168–72).
The development of NO-releasing polymers containing NO/nucleophile complexes has been reported by Smith et al., (1996) J. Med. Chem., 39:1148–56. These materials were capable of releasing NO for as long as five weeks in vitro and were able to limit smooth muscle cell proliferation in culture and to reduce platelet adherence to vascular graft materials in an arterio-venous shunt model. These materials show promise for numerous clinical applications where localized NO production would be desired, such as anti-thrombotic coating materials for catheters, but probably will not be useful for the direct treatment of tissues in vivo as these materials suffer from a number of disadvantages. These polymers may be produced as films, powders, or microspheres, but they cannot be formed in situ in direct contact with cells and tissues, thus making it difficult to strictly localize NO treatment to a tissue and potentially causing issues with the retention of the polymer at the site of application. The formulation issues will also make local administration during laparoscopic or catheter-based procedures difficult or impossible. Additionally, biocompatibility of the base polymer is a serious issue for implantable, NO-releasing polymers, especially those intended for long-term use, as inflammatory and thrombotic responses may develop after the cessation of NO release.
With respect to chronic wound healing, approaches that are common today are typically based on simple wound care regimens involving debridement, cleaning, and application of moist dressings (Thomas S, Leigh (1998). WOUND DRESSINGS. WOUNDS: BIOLOGY AND MANAGEMENT, D. Leaper and K. Harding. New York, N.Y., Oxford University Press). More advanced dressings such as topical gels containing growth factors have resulted in enhanced healing rates in some clinical studies (Wieman T, Smiell J, Su Y. Efficacy and safety of a topical gel formulation of recombinant human platelet-derived growth factor-BB (beclapermin) in patients with nonhealing diabetic ulcers: a phase III randomized, placebo-controlled, double-blind study. Diabetes Care 1998; 21: 822–827; Wieman T J and the Beclapermin Gel Studies Group. Clinical efficacy of Beclapermin (rhPDGF-BB) Gel. Am J Surg 1998; 176: 74S–79S; Martinez-de Jesus F R, Morales-Guzman M, Gastaneda M, Perez-Morales A, Garcia-Alsono J, Mendiola-Segura L. Randomized single-blind trial of topical ketanserin for healing acceleration of diabetic foot ulcers. Arch Med Res 1997; 28: 95–99), however on the whole these treatments are difficult to apply and are often too expensive for application to large, chronic wounds. Additionally, not all chronic wounds display growth factor deficiencies, and other mechanisms such as rapid degradation by wound proteinases may be involved in the reduction of growth factor levels observed in many chronic wounds. Many chronic wounds are unresponsive to growth factor therapy (Greenhalgh D. The role of growth factors, in wound healing. J Trauma 1996; 41: 159–167).
With respect to proliferation of endothelial cells, it has been shown that the presence of NO decreases endothelial cell proliferation. See, for example, Heller, R., Polack, T., Grabner, R., Till, U. (1999) “Nitric oxide inhibits proliferation of human endothelial cells via a mechanism independent of cGMP”, Atherosclerosis, 144:49–57; and Sarkar, R., Webb, R. C., Stanley, J. C. (1995) “Nitric oxide inhibition of endothelial cell mitogenesis and proliferation”, Surgery, 118:274–9. However, these studies utilized very high doses of NO-releasing drugs, which may account for the decreased endothelial cell proliferation. Additionally, previous researchers have found it difficult to seed endothelial cells onto devices: Scott-Burden, T., Tock, C. L., Schwarz, J. J., Casscells, S. W., Engler, D. A. (1996) “Genetically engineered smooth muscle cells as linings to improve the biocompatibility of cardiovascular prostheses”, Circulation, 94:235–8.
It is believed by the inventors that the development of materials that encourage the proliferation and/or migration of endothelial cells should enhance the growth of endogenous endothelial cells from tissue surrounding an implant onto the implant surface. Therefore, applicants propose that endothelialization of blood-contacting implants, such as stents, grafts, and ventricular assist devices, may significantly improve device performance by decreasing thrombogenicity and smooth muscle cell proliferation.
It would be more efficient if NO releasing compounds or compounds modulating NO levels could be administered solely to the site in need of treatment, and in some cases, reduce or eliminate side effects due to systemic administration of the agents, particularly over prolonged time periods.